KR100307069B1 - Inhibition of Engiogenin and Plasmin - Google Patents

Abstract

The present invention relates to inhibitors of angiogenin and plasmin. More specifically, the present invention relates to a chANG peptide (a gene codon corresponding to a base sequence encoding amino acids 58 to 68 of human engiogenin, reported as an inhibitor of angiogenin inducing renal angiogenesis. Peptide consisting of amino acids translated from anticodon (see patent application 97-44681) and specific deletion peptides derived therefrom and methods for inhibiting renal angiogenesis using the same The peptides of the present invention are renal angiogenesis and cancer cells It inhibits the activity of plasmin, which is essential for metastasis, and inhibits neovascularization induced by engiogenin and human cancer cells without affecting existing blood vessels. As a therapeutic agent for diseases such as rheumatoid arthritis, diabetic blindness and blindness due to macular degeneration of the retina Can be used effectively.

Description

INHIBITOR OF ANGIOGENIN AND PLASMIN

The present invention relates to inhibitors of angiogenin and plasmin. More specifically, the present invention relates to a chANG peptide (a gene codon corresponding to a base sequence encoding amino acids 58 to 68 of human engiogenin, reported as an inhibitor of angiogenin inducing renal angiogenesis. peptides consisting of amino acids translated from anticodon (see patent applications 44681 and Gho, YS et. al., J. Biol. Chem., 272: 24294 (1997)) and specific deletion peptides derived therefrom (chANGN- 1, chANGN-3, and chANGN-5) are concentration-dependently inhibited the activity of plasmin, which is essential for renal angiogenesis and metastasis of cancer cells, and is directed to a method of inhibiting renal angiogenesis induced by human mammary gland cancer. will be.

Renal angiogenesis is a term used to describe the formation of new blood vessels in existing blood vessels, and is essential in normal conditions such as organ formation or wound healing, as well as in various diseases such as cancer cell growth and metastasis, rheumatoid arthritis and diabetic blindness. Is required.

Renal angiogenesis is regulated by the precise interaction between inducers and inhibitors of angiogenesis in normal conditions, but in many diseases, such as cancer cell growth and metastasis, rheumatoid arthritis and diabetic blindness, Will happen. In fact, in various types of human cancers, there is a close correlation between the pretreatment of capillaries formed in cancer tissues and the possibility of cancer metastasis.

Engiogenin (Fett, JW et. Al., Biochemistry, 24: 5480 (1985)) is one of several dozen known angiogenic inducers, which are essential for the growth and metastasis of cancer cells that secrete it. Known by Olson, KA et. Al., Proc. Natl. Acad. Sci., USA, 92: 442 (1995). Engiogenin is known to induce renal angiogenesis through binding to a receptor called actin on the surface of endothelial cells that make up blood vessels. Hu, GF et. Al., Proc. Natl. Acad. Sci., USA, 90: 1217 (1993).

Renal angiogenesis and metastasis of cancer cells have common characteristics such as breakdown of intervaginal vagina and invasion into surrounding tissues. In this process, various proteolytic enzymes (metalloprotease, plasminogen activator and plasmin) Etc.) is essential. Actin inhibits plasmin activity (see Lind, SE et. Al., J. Biol. Chem., 266: 5273 (1991)) but increases the activity of the plasminogen activator that activates the plasma. The activity of plasmin produced by plasminogen activator in plasminogen increases in the presence of actin (see Lind, SE et. Al., J. Biol. Chem., 266: 17673 (1991)). It is known that plasmin activity increases even more in the presence of actin alone when both and angiogenin are present simultaneously (see Hu, GF et. Al., Biochem. Biophys. Res. Commun., 197: 682 (1993). )). This is known to occur because a combination of actin and angiogenin increases the activity of plasmin produced by plasminogen activator in plasminogen without inhibiting the activity of plasmin. Therefore, the binding sites of the actin-binding enzyme and plasmin are expected to be structurally similar, and substances that bind to the actin-binding enzyme and inhibit the binding of actin, for example chANG peptides, may inhibit the activity of plasmin and cancer cells. It was also predicted that they could inhibit growth and metastasis.

On the other hand, most of the currently used cancer treatments have some effects on certain early cancers, but the cancer cells show rapid resistance to these anticancer agents and their types are very diverse, and they have little effect once metastasis occurs. As a result, many limitations have been exposed to clinical use. Therefore, there is an urgent need for the development of a new cancer treatment that can overcome the above problems.

Accordingly, the present inventors have studied to develop a neovascularization inhibitor that can be used regardless of the aforementioned problems, that is, the variety of cancer cell types, obtaining resistance to anticancer agents, and metastasis of cancer cells, and thus, binding to actin, a human engiogenin receptor. ChANG peptides and deletion peptides composed of amino acids translated from an anticodon corresponding to the nucleotide sequences encoding amino acids 58 to 68 of human engiogenin involved in renal angiogenesis and cancer cells Inhibits the activity of the enzyme and plasmin required in the process of tissue breakdown and invasion, which is essential for metastasis, and effectively inhibits the neovascularization induced by the enzyme and human mammary gland cancer. The present invention has been completed.

After all, the main object of the present invention is to provide human angiogenin that induces renal angiogenesis and peptides with antagonistic ability against plasmin which is essential for renal angiogenesis and metastasis of cancer cells.

Another object of the present invention is to provide a method of inhibiting renal angiogenesis by administering the electrical peptides to a mammal in which renal angiogenesis continues abnormally.

1 is a graph showing that plasmin inhibits the binding between human engiogenin and its receptor actin in a concentration-dependent manner.

2 is a graph showing that the chANG peptide acts as a noncompetitive inhibitor of plasmin.

3 is a graph showing that chANG peptides inhibit the invasion by concentration-dependent degradation of intercellular vaginal cells of human mammary gland cancer (fibrosarcoma).

4 is a graph showing that the chANG peptide and specific deletion peptides derived therefrom (chANG-1, chANGN-3 and chANGN-5) concentration-dependently inhibit the activity of plasmin.

5 is a graph showing that renal angiogenesis induced by human engiogenin is inhibited by chANG peptides and specific deletion peptides derived from them (chANG-1, chANGN-3 and chANGN-5).

FIG. 6 is a graph showing that renal angiogenesis induced by human mammary cancer is inhibited by chANG peptide and certain deletion peptides derived from it (chANGN-1, chANGN-3 and chANGN-5).

Hereinafter, the present invention will be described in detail.

Actin inhibits the activity of plasmin, a protease required for renal angiogenesis and metastasis of cancer cells (see Lind, SE et al., J. Biol. Chem., 266: 5273 (1991)). The combination of and angiogenin has not been reported to inhibit the activity of plasmin (Hu, GF et. Al., Biochem. Biophys. Res. Commun., 197: 682 (1993), with angiogenin binding to actin). The binding site of plasmin is expected to be structurally similar, and it is expected that a substance that binds to angiogenin and inhibits actin binding may inhibit plasmin activity and inhibit cancer cell growth and metastasis.

Based on this report, the present inventors found that the chANG peptide (anticodon corresponding to the base sequence encoding amino acids 58 to 68 of human engiogenin, reported as an inhibitor of angiogenin inducing renal angiogenesis). Peptides consisting of amino acids translated from (see patent applications 44681 and Gho, YS et. Al., J. Biol. Chem., 272: 249294 (1997)) and deletion peptides derived therefrom were chemically synthesized.

chANG: NH ₂ -Val-Phe-Ser-Val-Arg-Val-Ser-Ile-Leu-Val-Phe-COOH

chANGN-1: NH ₂ --Phe-Ser-Val-Arg-Val-Ser-Ile-Leu-Val-Phe-COOH

chANGN-3: NH ₂ -Val-Arg-Val-Ser-Ile-Leu-Val-Phe-COOH

chANGN-5: NH ₂ -Val-Ser-Ile-Leu-Val-Phe-COOH

Among these peptides, the chANG peptides non-competitively inhibit the activity of plasmin (noncompetitive inhibitor) and concentration-dependently inhibit the invasion by intercellular vaginal degradation of human mammary gland cancer. In addition, chANG peptides and specific deletion peptides derived from them (chANG-1, chANG-3 and chANG-5) inhibit the activity of plasmin and inhibit renal angiogenesis induced by engiogenin and human mammary gland cancer. Since it does not affect blood vessels, it may be usefully used as a cancer drug without side effects.

Therefore, the peptides of the present invention can suppress renal angiogenesis by administering to a mammal with a disease or the like in which renal angiogenesis is abnormally persisted or to a mammalian model exhibiting electrical symptoms.

On the other hand, it is apparent that peptide derivatives prepared by adding site-specific mutations such as substitution, deletion and addition of amino acids to the peptides of the present invention also belong to the scope of the present invention. In addition, as well as the four types of peptides, as well as derivatives thereof, inhibits the binding of human angiogenin to actin known as its receptor and inhibits the activity of plasmin, thus inhibiting only angiogenesis without affecting existing blood vessels. It may be effectively used as a therapeutic agent for cancer or for diseases such as rheumatoid arthritis, diabetic blindness, and blindness due to macular degeneration of the retina.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention in detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Design and Synthesis of Peptides Used in the Present Invention (FIG. 1)

ChANG peptides reported as inhibitors of angiogenin inducing renal angiogenesis for carrying out the invention (see patent applications 97-44681 and Gho, YS et. Al., J. Biol. Chem., 272: 24294 (1997) ) And the deletion peptides derived therefrom were chemically synthesized (see FIG. 1).

In FIG. 1, the chANG peptide is composed of amino acids translated from an anticodon corresponding to a nucleotide sequence encoding amino acids 58 to 68 which are involved in binding to actin known as its receptor in human engiogenin. The chANG derivatives were designed by sequentially removing amino acids at the amino terminus (chANG-1, chANG-3 and chANG-5) or at the carboxy terminus (chANG-2 and chANG-4) at chANG.

The peptides designed above were chemically synthesized (Bio-Synthesis, Inc., USA), then the synthesized peptides were injected into a C18 reverse phase column for high performance liquid chromatography, solvent A (distilled water / 0.05% trifluoroacetic acid) and solvent Purification using B (acetonitrile / 0.05% trifluoroacetic acid). Purified peptides were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mg / ml, dispensed in appropriate quantities and stored at -70 ° C.

Example 2 Assay for Inhibiting Binding Between Plasmin of Human Engiogenin and Actin, Its Receptor (FIG. 2)

Actin inhibits the activity of plasmin, a protease required for renal angiogenesis and metastasis of cancer cells (Lin, SE et al., J. Biol. Chem., 266: 5273 (1991)). In the presence of plasmin activity (Hu, GF et. Al., Biochem. Biophys. Res. Commun., 197: 682 (1993)). Was predicted to be structurally similar. To test this, the following experiments were performed: 100 ng of angiogenin was immobilized in a 96-well ELISA container, phosphate buffer containing 1% albumin was added, and left at room temperature for 2 hours. Then, biotin-labeled actin (8.0 nM) was reacted with various concentrations of plasmin at room temperature for 30 minutes (at this time, aprotinin was added to inhibit the proteolytic ability of plasmin). (1) added to the vessel and fixed with angiogenin at room temperature for 1 hour, unbound actin was washed three times with 0.1% Tween 20 phosphate buffer solution and diluted to 1 / 5,000 peroxidase. The prepared streptavidin was treated at room temperature for 1 hour, and then washed three times with phosphate buffer containing 0.1% of Tween 20, followed by O-phenyldiaminedihydrochloride (OPD), an aqueous substrate of peroxidase. After a period of time, the reaction was stopped with 3N hydrochloric acid and the absorbance was measured at a wavelength of 490 nm to determine the amount of actin bound to angiogenin.

As a result, plasmin inhibited concentration-dependently the binding between human angiogenin and its receptor actin (see Fig. 2). In Example 2 above, it was confirmed that the binding sites of the enzyme and plasmin binding to actin are structurally similar.

Example 3: Assay of inhibitory ability of plasmin activity of chANG peptide (FIG. 3)

In order to determine whether the purified chANG peptide synthesized in Example 1 inhibits the activity of plasmin as follows. 0.2 ml of a solution containing plasmin (1 μg) and its substrate, ChromoPL and various concentrations of chANG peptides (20 mM Tris / HCI, pH 7.5, 130 mM NaCl) were added to a 96-well ELISA vessel and reacted at room temperature at 490 nm. Absorbance was measured at the wavelength. The activity of plasmin was determined by the initial activation rate (v), which was expressed as the change in absorbance at 490 nm wavelength per minute.

As a result, it was shown that chANG peptides inhibited plasmin activity in a concentration-dependent manner, and chANG peptides acted as noncompetitive inhibitors of plasmin (see FIG. 3).

Therefore, the chANG peptide was expected to inhibit renal angiogenesis and cancer cell metastasis by inhibiting plasmin activity required for renal angiogenesis and cancer cell metastasis.

Example 4 Inhibition of Invasion Ability by Intracellular Vaginal Degradation of Human Mammary Cancer of chANG Peptides

To determine whether chANG peptides inhibit cancer cell invasion, the following liver experiments were performed using a Biocoated Matrigel Invasion Chamber (Becton Dickinson, USA). 3T3 conditioned medium was added to the bottom of the invasion chamber and 0.2 ml of culture medium containing 100,000 human mammary cancer (HT1080, human fibrosarcoma) cells, 0.1% albumin and various concentrations of chANG peptides (DMEM) Was added to the top of the invasion chamber. After 16 hours of incubation at 37 ° C., cancer cells remaining in the upper filter were removed with a cotton swab and the number of cancer cells invaded.

As a result, the chANG peptide inhibited the concentration of human cancer cells in a concentration dependent manner. (See FIG. 4). In addition, alpha2 antiplasmin, known as an inhibitor of plasmin, also inhibited the invasion of human cancer cells. This suggests that the chANG peptide may be useful as a therapeutic agent that inhibits the invasion of cancer cells by inhibiting the activity of plasmin and thereby inhibits renal angiogenesis and metastasis of cancer cells.

Example 5 Assay for Plasmin Activity Inhibition of chANG Peptides and Deletion Peptides Derived therefrom (FIG. 5)

From Examples 3 and 4 above, it was found that the chANG peptides are noncompetitive inhibitors of plasmin activity (noncompetitive inhibitor) and inhibit the invasion of cancer cells by this mechanism. Although the chANG peptide is a relatively small substance consisting of 11 amino acids, the smaller the size, the more useful as a therapeutic agent for cancer from various viewpoints. Thus, various types of deletion peptides shown in Example 1 are synthesized and purified, and these peptides are used for the activity of plasmin. In order to determine whether to inhibit the experiment in the same manner as in Example 3.

As a result, specific deletion peptides (chANGN-1, chANGN-3 and chANGN-5) concentration-dependently inhibited plasmin activity, and chANGN-6, chANGN-2 and chANGN-4 peptides had no effect on plasmin activity. Was absent (see Figure 5). In particular, chANGN-5 (NH2-Val-Ser-Ile-Leu-Val-Phe-COOH) consisting of 6 amino acids inhibited plasmin activity more effectively than the chANG peptide consisting of 11 amino acids.

Example 6: Retinal Angiogenesis Inhibition Assay Induced by Engiogenin of chANG Peptide and Deletion Peptides Derived therefrom (FIG. 6)

Whether or not renal angiogenesis induced by angiogenin was inhibited by chANG peptides and deletion peptides derived from it was examined through experiments using an egg chorioallantoic membrane (CAM). In other words, Yoo Jeong-Ran (Pulmuone Co., Ltd.) was placed in an incubator at 37 ° C. for 2 days, 3 ml of albumin was removed, and 3 cm x 3 cm of egg shell was removed 4 days later. . Mix 10 ng of geogenin (0.69 pmol) and 500 ng of human albumin with a certain amount of chANG peptides and deletion peptides derived therefrom, 3 μl of the aliquots were added to 1/4 piece of Thermanox disc (Nunc, USA) and dried. The eggs were incubated on CAMs for 9 days, incubated for 2 days, and determined if there were blood vessels leading to the instilled samples. The experiment was conducted using 12-16 eggs per each sample and repeated three times.

As shown in FIG. 6, 10 ng (0.69 pmol) of engiogenin induced renal angiogenesis in 57.8% of eggs when present with 500 ng of human albumin (Angiogenin dashed line), but only 500 ng of human albumin was 38.8% Only eggs were induced renal angiogenesis (HSA dashed line). This indicates that engiogenin induces renal angiogenesis even in the presence of human albumin. In addition, chANG peptides that inhibit the activity of plasmin and specific deletion peptides derived from them (chANGN-1, chANGN-3, chANGN-5) may inhibit the activity of plasmin which is required for renal angiogenesis and metastasis of cancer cells. In addition, it was found that they effectively inhibit renal angiogenesis induced by engiogenin.

Example 7: A test for inhibition of renal angiogenesis induced by human cancer cells of chANG peptides and deletion peptides derived therefrom (FIG. 7)

Experiments with egg CAM examined whether chANG peptides and deletion peptides derived from them inhibit renal angiogenesis induced by human cancer cells. In the same manner as in Example 6, the fertilized egg was incubated at 37 ° C. to remove albumin and to make a window. 10 ⁵ human mammary gland cancers (HT1089) and 7.5 μg of Rat tail Becton Dickinson, USA, peptides were mixed in a volume of 5 μl, dipped into 1/4 piece of Thermanox disc and dried, and the collagen sponge at room temperature. After the preparation, it was placed on the CA of eggs incubated for 10 days and incubated for 3 days to determine whether there were blood vessels induced by the instilled sample. The experiment was carried out using 10-12 eggs per sample and repeated twice.

As a result, as shown in FIG. 7, in the presence of cancer cells, 72.78% of the eggs induced renal angiogenesis (FIG. 7, HT1080 dotted line), but collagen only induced renal angiogenesis only in 22.8% of the eggs (FIG. 7). , Collagen dotted line). This suggests that cancer cells induce renal angiogenesis toward the collagen sponge in the surrounding vessels. In addition, chANG peptides that inhibit the activity of plasmin and certain deletion peptides derived from them (chANGN-1, chANGN-3 and chANGN-5) are induced by human mammary cancer (HT1080) without affecting existing blood vessels. Renal angiogenesis was inhibited concentration dependently. However, chANGN-6, chANGN-2 and chANGN-4 peptides that did not affect plasmin activity had no effect on renal angiogenesis induced by cancer cells.

Thus, the chANG peptide and certain deletion peptides derived from it (chANGN-1, chANGN-3 and chANGN-5) not only inhibit the activity of plasmin that is required in renal angiogenesis and metastasis of cancer cells, but also in It effectively inhibited renal angiogenesis induced by cancer cells, but did not affect existing blood vessels, and thus could be usefully used as a cancer treatment agent without side effects.

As described and demonstrated in detail above, the peptides of the present invention inhibit the activity of plasmin, which is essential for neurogenesis and metastasis of cancer cells, and effectively inhibit the neovascularization induced by engiogenin and human cancer cells. Since it does not affect blood vessels, it may be usefully used as a therapeutic agent for diseases such as rheumatoid arthritis, diabetic blindness, and blindness due to macular degeneration of the retina, which require an anticancer agent or renal angiogenesis.

[SEQ ID NO: 1]

Claims (7)

Peptide consisting of the following amino acid sequence that inhibits the activity of plasmin to inhibit renal angiogenesis. NH ₂ -Val-Phe-Ser-Val-Arg-Val-Ser-Ile-Leu-Val-Phe-COOH Peptide consisting of the following amino acid sequence to inhibit the activity of plasmin to inhibit renal angiogenesis (angiogenesis). NH ₂ -Phe-Ser-Val-Arg-Val-Ser-Ile-Leu-Val-Phe-COOH Peptide consisting of the following amino acid sequence to inhibit the activity of plasmin to inhibit renal angiogenesis (angiogenesis). NH ₂ -Val-Arg-Val-Ser-Ile-Leu-Val-Phe-COOH Peptide consisting of the following amino acid sequence to inhibit the activity of plasmin to inhibit renal angiogenesis (angiogenesis). NH ₂ -Val-Ser-Ile-Leu-Val-Phe-COOH The peptide according to any one of claims 1, 2, 3 or 4, wherein the inhibition of the invasion by degrading the intercellular vaginal of human cancer cells inhibits the plasmin activity of the peptide. The peptide of any one of claims 1, 2, 3 or 4, which inhibits renal angiogenesis induced by angiogenin and human cancer cells without affecting existing blood vessels. The peptide of any one of claims 1, 2, 3 or 4 as a method of inhibiting renal angiogenesis comprising administering to a mammal in which renal angiogenesis is abnormally sustained.